Thrombin enzymatically released two fibrinopeptides from citraconylated canine fibrinogen. The rate of removal of modified fibrinopeptide-A was increased 40% over the rate of release of peptide-A from native canine fibrinogen. The modification of 80 or more lysine groups inhibited peptide release which was attributed to a conformational change in the structure of fibrinogen. Edman analysis verified the presence of a modified lysine in fibrinopeptide-A. The peptide release from human fibrinogen, a species lacking lysine in peptide-A, was not altered by modification. On the other hand, the rate of release of bovine fibrinopeptide-B by thrombin was increased by citraconylation of the lysine residue in the peptide. The presence of a carboxylic acid residue in the 6th position from the arginylglycyl bond cleaved by thrombin influences the rate of release of the peptides. Citraconylation of fibrinogen can be reversed without disturbing the periodicity obtained during polymer formation. Radioactive glycine ethyl ester incorporated glycine residues into fibrinogen, revealing that 4 of the 5 carboxylic acid residues in fibrinopeptide-A were modified during methylation. The glutamic acid residue in the 6th position was implicated in the reduction of thrombin activity toward methylated fibrinogen.